ABSTRACT The overall goal of this project from Koutif Therapeutics (Koutif) is to develop a novel class of oral small molecule compounds, inhibitors of Fbxo3 (an E3 ligase), for the treatment of Crohn?s disease (CD). CD involves chronic inflammation of the gastro-intestinal tract, and has a multifactorial pathogenesis, involving genetic, environmental, and microbial inputs, several cytokines, and dysfunctional responses from the innate and adaptive immune systems. Despite numerous approved medicines for CD, only about 50% of patients respond to treatment, with only about 10% experiencing full remission, and there is a high rate of relapse over time. There are safety concerns with current drugs, including the risk of opportunistic infections and malignancies, further highlighting the large unmet medical need in CD. In this application Koutif plans to translate previous pioneering work by its scientific founders, Drs. Rama Mallampalli and Bill Chen, to develop therapeutic compounds that inhibit the inflammatory cascade in a unique manner, involving the targeting of Fbxo3, an E3 ligase subunit, which is a central regulator of pro-inflammatory cytokine production. Fbxo3 increases the release of inflammatory mediators by degrading an anti-inflammatory protein, called Fbxl2. Fbxl2, also an E3 ligase, is a sentinel inhibitor of the TRAFs which control the release of various cytokines, including TNF-? and IL-6, via the NF-?B signaling pathway. Fbxo3 and Fbxl2 also regulate the inflammasome, NLRP3, which mediates IL-1? and IL-18 release. Studies by our founders identified specific inhibitors of Fbxo3 that degrade several potent, pro-inflammatory proteins, and decreased multiple disease-relevant cytokines involved in adaptive and innate signaling pathways. Koutif?s lead compound, BC-1261, has reached clinical candidate status to be evaluated for the treatment of CD, after successfully completing IND-enabling studies. The proposed studies will examine the effects of BC-1261 in two preclinical models of CD, followed by Phase Ia clinical trial. The specific aims of this proposal are: 1) Expanded target validation for the use of BC-1261 in CD by i) examining its dose-response effects in two murine animal models (chronic DSS and SAMP/YitFc spontaneous ileitis) and ii) comparing the expression and cellular sources of BC-1261 molecular targets (Fbxo3, Fbxl2, TRAFs and NLRP3) in intestinal specimens from IBD patients (both CD and UC) compared to control subjects; and 2) Phase 1a single ascending oral dose study to test the PK, PD, and safety of BC-1261 in healthy subjects. The long-term project goal is to develop oral BC-1261, a member of a novel class of compounds (Fbxo3 inhibitors) as a more effective and safer medicine than current treatments for CD.